1. Field of the Invention
The present invention relates generally to the diagnosis of cancer. The invention concerns the creation of probes for use in diagnosing and monitoring certain genetic abnormalities, including those found in leukemia and lymphoma, using molecular biological hybridization techniques. In particular, it concerns the localization of the translocation breakpoint on the MLL gene, the identification of nucleic acid probes capable of detecting rearrangements in all patients with the common 11q23 translocations and the identification of MLL mRNA transcripts characteristic of leukemic cells. MLL fusion proteins and anti-MLL antibodies are also disclosed.
2. Description of the Related Art
The etiology of a substantial portion of human diseases lies, at least in part, with genetic factors. The identification and detection of genetic factors associated with particular diseases or malformations provides a means for diagnosis and for planning the most effective course of treatment. For some conditions, early detection may allow prevention or amelioration of the devastating courses of the particular disease.
The genetic material of an organism is located within one or more microscopically visible entities termed chromosomes. In higher organisms, such as man, chromosomes contain the genetic material DNA and also contain various proteins and RNA. The study of chromosomes, termed cytogenetics, is often an important aspect of disease diagnosis. One class of genetic factors which lead to various disease states are chromosomal aberrations, i.e., deviations in the expected number and/or structure of chromosomes for a particular species or for certain cell types within a species.
There are several classes of structural aberrations which may involve either the autosomal or sex chromosomes, or a combination of both. Such aberrations may be detected by noting changes in chromosome morphology, as evidenced by band patterns, in one or more chromosomes. Normal phenotypes may be associated with rearrangements if the amount of genetic material has not been altered, however, physical or mental anomalies result from chromosomal rearrangements where there has been a gain or loss of genetic material. Deletions, or deficiencies, refer to loss of part of a chromosome, whereas duplication refers to addition of material to chromosomes. Duplication and deficiency of genetic material can be produced by breakage of chromosomes, by errors during DNA synthesis, or as a consequence of segregation of other rearrangements into gametes.
Translocations are interchromosomal rearrangements effected by breakage and transfer of part of chromosomes to different locations. In reciprocal translocations, pieces of chromosomes are exchanged between two or more chromosomes. Generally, the exchanges of interest are between non-homologous chromosomes. If all the original genetic material appears to be preserved, this condition is referred to as balanced. Unbalanced forms have duplications or deficiencies of genetic material associated with the exchange; that is, some material has been gained or lost in the process.
One of the most interesting associations between chromosomal aberrations and human disease is that between chromosomal aberrations and cancer. Non-random translocations involving chromosome 11 band q23 occur frequently in both myeloid and lymphoblastic leukemias (Rowley, 1990b; Heim & Mitelman, 1987). The four most common reciprocal translocations are t(4;11) and t(11;19), which exhibit mainly lymphoblastic markers and sometimes monocytic markers, or both lymphoblastic and monoblastic markers; and t(6;11) and t(9;11), which are mainly found in monoblastic and/or myeloblastic leukemias (Mitelman et al., 1991). Other chromosomes which are involved in recurring translocations with this band in acute leukemias are chromosomes X, 1, 2, 10, and 17.
The present inventors have previously demonstrated, by fluorescence in situ hybridization (FISH), that a yeast artificial chromosome (YAC) containing the CD3D and CD3G genes was split in cells with the four most common translocations (Rowley et al., 1990). Further studies led the inventors to the identification of the gene located at the breakpoint, which was named MLL for mixed lineage leukemia or myeloid/lymphoid leukemia (Ziemin-van Der Poel et al., 1991). The MLL gene has also been independently termed ALL-1 (Cimino et al., 1991; Gu et al., 1992a; b), Htrx (Djabali et al., 1992) and HRX (Tkachuk et al., 1992). The present inventors differentiated the more centromeric MLL rearrangements from the more telomeric breakpoint translocations which involve the RCK locus (Akao et al., 1991b) or the p54 gene (Lu & Yunis, 1992).
From the same YAC clone as described by the present inventors (Rowley et al., 1990), a DNA fragment was obtained which allowed the detection of rearrangements in leukemic cells from certain patients (Cimino et al., 1991; 1992). This 0.7 kilobase DdeI fragment allowed detection of rearrangements in a 5.8 kilobase region in 6 of 7 patients with the t(4;11), 4 of 5 with t(9;11), and 3 of 4 with the t(11;19) translocations (Cimino et al., 1992). Combining these results with those from a subsequent series including an additional 14 patients, the DdeI fragment probe was found to detect rearrangements in 26 of 30 cases with t(4;11), t(9;11) and t(11;19) translocations (Cimino et al., 1991; 1992), which represents an overall detection rate of 87%. Despite this partial success, the failure of the DdeI probe to detect all rearrangements is a significant drawback to its use in clinical diagnosis.
Accordingly, prior to the present invention, there remained a particular need for the identification of nucleic acid fragments or probes capable of detecting leukemic cells from all patients with the common 11q23 translocations. The creation of such probes which may be used in both Southern blot analyses and in FISH with either dividing leukemic cells or interphase nuclei would be particularly important. The elucidation of further information regarding the MLL gene, such as further sequence data and information regarding transcription into mRNA, would also be advantageous, as would the identification of nucleic acid fragments capable of differentiating MLL mRNA transcripts from normal and leukemic cells.